Effect of ACTH and CRH on Plasma Levels of Cortisol and Prostaglandin F2α Metabolite in Cycling Gilts and Castrated Boars

The present study was designed to evaluate the effects of synthetic ACTH (1–24, tetracosactid) and porcine CRH on the plasma levels of cortisol and PGF2α metabolite in cycling gilts (n = 3) and castrated boars (n = 3). The experiments were designed as crossover studies for each gender separately. Each animal received, during three consecutive days; 1) ACTH (Synacthen® Depot) at a dose of 10 μg/kg body weight in 5 ml physiological saline, 2) porcine CRH at a dose 0.6 μg/kg body weight in 5 ml physiological saline or 3) physiological saline (5 ml). The test substances were administered via an indwelling jugular cannula in randomized order according to a Latin square. The administration of ACTH to cycling gilts resulted in concomitant elevations of cortisol and PGF2α metabolite with peak levels reached at 70.0 ± 10.0 and 33.3 ± 6.7 min, respectively. Similarly, the administration of ACTH to castrated boars resulted in concomitant elevation of cortisol and PGF2α metabolite with peak levels reached at 60.0 ± 0.0 and 20.0 ± 0.0 min, respectively. Cortisol peaked at 20 min after administration of CRH in both cycling gilts and castrated boars with maximum levels of 149.3 ± 16.5 nmol/1 and 138.3 ± 10.1 nmol/1, respectively. It can be concluded that administration of synthetic ACTH (tetracosactid) to pigs caused a concomitant elevation of cortisol and PGF2α metabolite levels in both cycling gilts as well as castrated boars. The administration of CRH to pigs resulted in an elevation of cortisol levels in both cycling gilts and castrated boars. Conversely, PGF2α metabolite levels were not influenced by the administration of CRH either in cycling gilts or in castrated boars.


Introduction
Corticotropin releasing hormone (CRH) plays a central role in regulating the release of adrenocorticotropic hormone (ACTH) during a stress response. ACTH acts on the adrenal glands, inducing the secretion of cortisol. In our previous study, we reported that ACTH administration to ovariectomized gilts results in the plasma elevation of cortisol, progesterone and prosta-glandin F 2α metabolite (Mwanza et al. 2000b). How ACTH is capable of stimulating the secretion of PGF 2α metabolite remains unanswered. However, it was previously suggested by Laychok & Rubin (1975) that ACTH enhances the conversion in vitro of 3 H-arachidonic acid to prostaglandins in feline adrenocortical cells. The findings of Anthonisen et al. (1997) indi-cate that prostaglandins in the brain interact in their stimulatory regulation of ACTH secretion. Such an interaction may also be involved in prostaglandins mediation of the ACTH response to immunochallenges. Abraham et al. (1998) reported that stimulation of porcine pituitary cells by relatively low concentrations of prostaglandin E 2 support increased secretion of ACTH but exposure to greater concentrations of this prostaglandin in fact suppresses ACTH secretion. Food deprivation, which is a form of stress, has been shown to result in the plasma elevation of both cortisol and PGF 2α metabolite (Tsuma et al. 1996;Mburu et al. 1998;Mwanza et al. 2000a;Razdan et al. 2001). Intracerebroventricular as well as intravenous injections of CRH resulted in an increased plasma cortisol concentration in pigs (Sakumato et al. 2004;Lang et al. 2004). Previously, it was suggested that CRH may also act directly or indirectly to enhance cortisol secretion beyond the level achieved through adrenal stimulation by ACTH (Minton & Parsons 1993). The objectives of the present study were to evaluate the effects of synthetic ACTH (tetracosactid) and porcine CRH on the plasma levels of cortisol and PGF 2α metabolite in cycling gilts and castrated boars.

Animals
Six crossbred pigs (Landrace x Yorkshire; three gilts and three castrated boars) aged approximately 6 months weighing between 110 and 125 kg were used for this experiment. The pigs were brought to the Division of Comparative Reproduction, Obstetrics and Udder Health, and were housed in individual pens. The stable had a light period of 12 h starting from 06:30 h and the room temperature varied between 20 and 23°C. The pigs were fed according to the Swedish breeding stock standard (Simonsson 1994). The gilts were stimulated by boars in the vicinity and were expected to come in oestrus within the first week after arrival. After the second oestrus, the experiments were carried out in the early luteal phase (days 5-10). The gilts were checked twice daily at 06:00 h and 18:00 h for signs of oestrus in the presence of a fertile boar by back-pressure test. All the six animals were vein-cannulated (Rodriguez & Kunavongkrit 1983) at about one week before the experiments. The experiments were designed as crossover studies for each gender separately. Each animal received, during three consecutive days; 1) ACTH (1-24) (Synacthen ® Depot, Novartis Pharma AG, Basel, Schweiz) at a dose of 10 µg/kg body weight in 5 ml physiological saline, 2) porcine CRH (American Peptide Company, Inc., Sunnyvale, CA, USA) at a dose 0.6 µg/kg body weight in 5 ml physiological saline or 3) physiological saline (5 ml). The test substances were administered via an indwelling jugular cannula in randomized order according to a Latin square. On the day of the experiment, blood samples were taken at -40, -20 min and immediately before injection. Treatment was performed at 10:00 h (time = 0) and blood samples were taken 20, 40,60,80,100,120,140,160,180,210,240,270,300,330 and 360 min after injection. Blood was collected in ten ml heparinised tubes, centrifuged immediately and plasma stored at -20°C until analysed. The care of the animals and the experimental design of this study were approved by the Local Animal Ethics Committee in Uppsala, Sweden.

Hormone assays
Cortisol. Plasma cortisol was determined by radioimmunoassay (Coat-A-Count Cortisol, Diagnostic Products Corporation, Los Angeles, CA, USA) according to the manufacturer's instructions. Serial dilutions of porcine plasma with high concentrations of cortisol produced displacement curves parallel to the standard curve. The intra-assay coefficients of variation calculated from 5 assays were 22% at 14 nmol/l, 14% at 28 nmol/l and decreased below 8% for concentrations between 138 and 552 nmol/l. The inter-assay coefficients of variation for three control samples were 13% (33 nmol/l), 9% (74 nmol/l) and 9% (541 nmol/l). The average detection limit of the assay was 7 nmol/l. Prostaglandin F 2α metabolite. The main initial blood plasma metabolite of prostaglandin F 2α , 15-keto-13,14-dihydro-PGF 2α (15-ketodihydro-PGF 2α ), was analysed by radioimmunoassay according to Kunavongkrit et al. (1983). The relative cross-reactions of the antibody were 16% with 15-keto-PGF 2α , and 4% with 13,14dihydro-PGF 2α . The intra-assay coefficients of variation ranged between 3.4 and 7.6% for different ranges of the standard curve and the inter-assay coefficient of variation was around 14%. The practical limit of sensitivity for the assay analyzing 0.2 ml of plasma was 60 pmol/l.

Statistical analyses
Data were examined by analysis of variance using MIXED procedure according to SAS package (Statistical Analysis Systems 1989). Additionally, the area under the curve, peak value, time and duration of the peak were calculated for each animal according to the GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). Data are expressed as means ± S.E.M. Probabilities less then 0.05 were considered significant.

Results
No significant (P>0.05) differences were seen in the pretreatment plasma levels of cortisol or PGF 2α metabolite between the saline, ACTH and CRH treated cycling gilts (Figures 1 and 2). The administration of ACTH to cycling gilts resulted in concomitant elevations of cortisol ( Figure 1) and PGF 2α metabolite ( Figure 2) with peak levels reached at 70.0 ± 10.0 and 33.3 ± 6.7 min, respectively. The durations of the peaks were 153.3 ± 28.2 and 103.2 ± 11.4 min, respectively and their maximum concentrations were 270.7 ± 16.5 nmol/l and 1517.7 ± 137.2 pmol/l, respectively. No significant (P>0.05) differences were seen in the pretreatment plasma levels of cortisol or PGF 2α metabolite between the saline, ACTH and CRH treated castrated boars (Figures 3 and  4). The administration of ACTH to castrated boars resulted in concomitant elevation of cortisol ( Figure 3) and PGF 2α metabolite ( Figure  4) with peak levels reached at 60.0 ± 0.0 and 20.0 ± 0.0 min, respectively. The durations of these peaks were 199.1 ± 30.0 and 86.3 ± 13.8 min, respectively and their maximum concentrations were 289.0 ± 10.1 nmol/l and 1262.3 ± 53.2 pmol/l, respectively. The administration of CRH to both cycling gilts and castrated boars resulted in the cortisol peak 20 min later with maximum levels of 149.3 ± 16.5 nmol/l ( Figure 1) and 138.3 ± 10.1 nmol/l (Figure 3), respectively. The durations of these peaks were 57.3 ± 18.5 min and 255.2 ± 43.6 min, respectively. Prostaglandin F 2α metabolite levels were not influenced by the injection of CRH either in cycling gilts or castrated boars (Figures 2 and 4). Physiological saline did not alter significantly either cortisol or PGF 2α metabolite levels in any animal (Figures 1-4). No significant (P>0.05) differences were seen in the measured responses between females and males.

Discussion
The present study clearly demonstrates that cortisol reach peak levels much lower and earlier in CRH (approximately after 20 min) than in ACTH (approximately after 70 min) treated cycling gilts or castrated boars.   approximately 30 min after administration of CRH and approximately 60-90 min after administration of synthetic ACTH to dairy cows. The present study also demonstrates that the administration of ACTH stimulates a concomitant elevation of both cortisol and PGF 2α metabolite levels in both cycling gilts and castrated boars. In addition, peak PGF 2α metabolite levels occur earlier than peak cortisol levels. Apparently, it takes approximately twice the time for cortisol than for PGF 2α metabolite to reach peak levels following ACTH administration. This is consistent with our previous findings in ovariectomized gilts (Mwanza et al. 2000b) and suggests that ACTH stimulates the secretion of PGF 2α earlier than cortisol. The frequency of blood collection may have impact on the occurrence of PGF 2α metabolite peak in relation to cortisol peak. When blood samples were taken only at 1-h intervals, both PGF 2α metabolite and cortisol peak were seen one hour after ACTH administration in recently ovulated sows (Razdan et al. 2002). (1994) have demonstrated that daily administration of ACTH from day 13 to day 16 of the oestrous cycle in multiparous Welsh Mountain ewes suppressed the levels of PGF 2α metabolite. They further showed that in ovariectomized multiparous Welsh Mountain ewes, primed first with progesterone and then with oestradiol-17ß, ACTH reduced the ability of oxytocin to cause the release of PGF 2α into the peripheral circulation. However, there is evidence that feline and rat adrenocortical cells synthesise prostaglandins F 2α and E 2 and that the total prostaglandins synthesis is stimulated by ACTH (Laychock & Rubin 1976;Chanderbhan et al. 1979). Winter et al. (1990 demonstrated that in vitro, the cytokine interleukin-1 enhances the conversion of 3 H-arachidonic acid to prostaglandins by cultured bovine adrenal cells. The secreted prostaglandins i.e. PGD 2 , PGF 2α and PGE 2 were in turn found to stimu-late cortisol secretion. Furthermore, Nashuhita et al. (1997) reported that intravenously administered PGE 1 , PGE 2 or PGF 2α had significant ACTH-releasing activity in the rat and suggested that prostaglandins are playing a role in regulating the hypothalamo-pituitary-adrenal axis. In sows, injection of PGF 2α after ovulation resulted in a dramatic cortisol elevation, which lasted approximately 1.5 h .